Stem Cell Engineering Group, University of Bonn - Life and Brain Center and Hertie Foundation, Sigmund-Freud Strasse 25, Bonn, Germany.
Stem Cells. 2010 May;28(5):894-902. doi: 10.1002/stem.417.
Combined application of DNA recombinases Cre and FLP enables tightly controlled independent and/or sequential gene regulations. However, in practice, such dual recombinase strategies are hampered by the comparably low efficiency of the FLP recombinase. Here, we present the engineering of a recombinant cell-permeant FLP protein (TAT-FLP) that induces recombination in >75% of fibroblasts and mouse as well as human embryonic stem (ES) cells. We show that TAT-FLP ideally complements the strength of cell-permeant Cre recombinase for genetic engineering as exemplified by FLP-ON-Cre-OFF, an inducible transgene expression cassette that enables tightly controlled expression in a reversible manner. We exemplify this concept by conditional overexpression of LacZ and the caudal-related homeobox transcription factor CDX2. We expect our FLP transduction system to become widely useful for numerous genetic interventions addressing complex biological questions and the generation of transgene-free therapeutically applicable ES cell-derived cells.
DNA 重组酶 Cre 和 FLP 的联合应用可实现严格控制的独立和/或连续基因调控。然而，在实践中，这种双重组酶策略受到 FLP 重组酶效率相对较低的限制。在这里，我们构建了一种重组细胞通透性 FLP 蛋白（TAT-FLP），它可诱导 >75%的成纤维细胞以及小鼠和人胚胎干细胞（ES 细胞）发生重组。我们表明，TAT-FLP 理想地补充了细胞通透性 Cre 重组酶的优势，例如 FLP-ON-Cre-OFF，这是一种可诱导的转基因表达盒，可通过可逆方式实现严格控制的表达。我们通过条件过表达 LacZ 和尾相关同源盒转录因子 CDX2 说明了这一概念。我们预计，我们的 FLP 转导系统将广泛用于解决复杂生物学问题和生成无转基因的治疗性应用的 ES 细胞衍生细胞的众多基因干预中。
